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Confocal and multiphoton microscopy are powerful techniques to study morphology and dynamics in cells and tissue, if fluorescent labeling is possible or autofluorescence is strong. For non-fluorescent molecules, Coherent anti-Stokes Raman scattering (CARS) microscopy provides chemical contrast based on intrinsic and highly specific vibrational properties of molecules eliminating the need for labeling. Just as other multiphoton techniques, CARS microscopy possesses three-dimensional sectioning capabilities. Leica Microsystems has combined the CARS imaging technology with its TCS SP5 confocal microscope to provide several advantages for CARS imaging. For CARS microscopy, two picosecond near-infrared lasers are overlapped spatially and temporally and sent into the scanhead of the confocal system. The software allows programmed, automatic switching between these light sources for multi-modal imaging. Furthermore the Leica TCS SP5 can be equipped with a non-descanned detector which will significantly enhance the signal. The Leica TCS SP5 scanhead combines two technologies in one system: a conventional scanner for maximum resolution and a resonant scanner for high time resolution. The fast scanner allows imaging speeds as high as 25 images/per second at a resolution of 512x512 pixel. This corresponds to true video-rate allowing to follow processes at these time-scales as well as the acquisition of three-dimensional stacks in a few seconds. This time resolution is critical to study live animals or human patients for which heart beat and muscle movements lead to a blurring of the image if the acquisition time is high. Furthermore with the resonant scanhead the sectioning is truly confocal and does not suffer from spatial leakage. In summary, CARS microscopy combined with the tandem scanner makes the Leica TCS SP5 a powerful tool for three-dimensional, label-free imaging of chemical and biological samples in vitro and in vivo.

Confocal and multiphoton microscopy are powerful fluorescence techniques for morphological and dynamics studies of labeled elements. For non-fluorescent components, CARS (Coherent Anti-Stokes Raman Scattering) microscopy can be used for imaging various elements of cells such as lipids, proteins, DNA, etc. This technique is based on the intrinsic vibrational properties of the molecules. Leica Microsystems has combined CARS technology with its TCS SP5 II confocal microscope to provide several advantages for CARS imaging. The Leica TCS SP5 II combines two technologies in one system: a conventional scanner for maximum resolution and a resonant scanner for high time resolution. For CARS microscopy, two picosecond near-infrared lasers are tightly overlapped spatially and temporally and sent directly into the confocal system. The conventional scanner can be used for morphological studies and the resonant scanner for following dynamic processes of unstained living cells. The fast scanner has several advantages over other solutions. First, the sectioning is truly confocal and does not suffer from spatial leakage. Second, the high speed (29 images/sec @ 512x512 pixels) provides fast data acquisition at video rates, allowing studies at the sub-cellular level. In summary, CARS microscopy combined with the tandem scanner makes the Leica TCS SP5 II a powerful tool for multi-modal and three-dimensional imaging of chemical and biological samples. We will present our solution and show results from recent studies with the Leica instrument to illustrate the high flexibility of our system.

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